SUMMARY Homologous recombination is a template-dependent DNA repair process that underpins DNA replication and genome stability, and is essential for chromosome segregation during meiosis. The fundamental recombination reaction is the formation of Joint Molecule (JM) intermediates via homologous pairing and DNA strand-exchange between a broken chromosome end and a homologous template. JMs are matured and resolved in distinct ways to produce products with or without an associated crossover. This proposal will investigate the in vivo roles of factors that regulate the formation and resolution of JMs during meiosis. The general hypothesis is that specialized JM processing factors are subject to distinct spatial and temporal regulation. Assigning in vivo functions to specific JM processing factors and understanding how they interact during recombination remain challenging issues, which are complicated by overlapping/compensatory activities and the need for specialized assays to monitor molecular phenotypes. DNA assays to monitor the chemical steps of recombination in vivo will form the cornerstone of this investigation. There are three distinct aims: AIM1. To investigate the regulation of pro-crossover factors by post-translational modification. We have identified phosphorylation sites on three meiosis-specific factors that stabilize JMs and promote crossing-over. These factors are the DNA helicase, Mer3, the MutS complex related to DNA mismatch repair factors, and a SUMO E3 ligase, Zip3. In this, aim our goal is to understand the functional consequences of these phosphorylations and identify the responsible kinases. AIM2. To characterize factors that promote crossover-specific resolution of JMs. We have identified factors that promote biased resolution of JMs into crossovers. In this aim, we will further characterize these factors in order to: (i) identify activities and interactions required for crossng-over; (ii) demonstrate that specific factors act at the time of JM resolution; (iii) understand how polo-kinase, Cdc5, activates crossover-specific resolution. AIM3. To understand how the Smc5/6 complex regulates processing of JMs during meiosis. We have shown that the SMC-family complex, Smc5/6 regulates both the formation and resolution of JMs. We will address the idea that smc5/6 mutants form pathological JM structures that cannot be processed by resolving enzymes. We will also investigate the general theme that the temporally distinct roles of the Smc5/6 complex in JM formation and JM resolution are mediated through regulation of distinct partner proteins, with a focus on the DNA helicases Mph1 and Sgs1. Results obtained from these studies will be broadly relevant for understanding meiotic recombination in all organisms and will be especially important for discerning meiotic processes that make crossing-over and homolog disjunction incredibly accurate processes.